Chromatography is a set of techniques for separating a mixture into its constituents. Generally, in a liquid chromatography analysis, a pump system takes in and delivers a mixture of liquid solvents (and/or other fluids) to a sample manager, where a sample awaits injection into the solvents. The sample is the material under analysis. Examples of samples include complex mixtures of proteins, protein precursors, protein fragments, reaction products, and other compounds, to list but a few. The mobile phase comprised of a sample dissolved in a mixture of solvents (and/or other fluids), moves to a point of use, such as a separation column, referred to as the stationary phase. By passing the mobile phase through the column, the various components in the sample separate from each other at different rates and thus elute from the column at different times. A detector receives the separated components from the column and produces an output from which the identity and quantity of the analytes may be determined.
Temperature can influence the results of the analysis, affecting such properties as the separation performance of the column and the viscosity of a mobile phase. Forcing a liquid phase (i.e., relatively non-compressible) through a packed bed column causes an increase in mobile phase temperature because of frictional (i.e., viscous) heating. Because thermal energy can be dissipated only through the outer surface of the column, a radial temperature gradient is formed within the column, with a warmer region being near the center of the column.
When employing compressible mobile phases a similar phenomenon occurs. In this instance, at certain regions of the phase diagram, an inverse radial temperature gradient (cooler near the center of the column) forms, caused by Joule-Thompson cooling of the mobile phase as it decompresses along the length of the column. Accordingly, the mobile phase cools as it travels along the length of the column. Because a column oven holds the outside of the column at a consistent temperature, radial temperature gradients are most severe near the outlet of the column, (i.e., where the mobile phase is coldest relative to the column exterior temperature). In both instances of heating and cooling, the magnitude of the radial temperature gradient increases as the diameter of the stationary phase particles decreases.
The formation of on-column radial temperature gradients causes a decrease in chromatographic performance. Because density, solvating power, viscosity, and analyte diffusivity, to name just a few properties, all depend on mobile phase temperature, a radial temperature gradient results in changes in analyte mobility across the cross-section of the column. Changes in analyte mobility result in regions of the analyte (i.e., chromatographic) band travelling faster or slower through the column than the bulk of the analyte band. This heterogeneity in analyte velocity results in broadening of the analyte band and, therefore, in a reduction of chromatographic efficiency. Therefore, minimizing the effects of radial thermal gradients in a column can be important to the accuracy and reproducibility of the results.
In addition to the formation of radial thermal gradients in SFC, linear velocity of the mobile phase can increase along the length of the column, which can have a negative effect on peak width. The pressure drop along the column results in a reduction in the CO2 density. Because the mass flow rate is conserved, a drop in density results in an increase in mobile phase linear velocity. The increase in the linear velocity will result in moving toward less efficient regions of a van Deemter curve. This change in linear velocity, although not observed with relatively incompressible mobile phases, such as is used in liquid chromatography (LC), has been a reason suggested for not using sub-2 μm particles with compressible mobile phases, such as is used in SFC.